JP3858065B2 - Novel N-acetylchitooligosaccharide deacetylase and method for producing the same - Google Patents

Description

【００１２】

以上の菌学的性質からバージーズ・マニュアル・オブ・デターミネイティブ・バクテリオロジー第８版の分類基準に従って公知の菌株と比較した結果、グラム陰性桿菌で、極毛性の鞭毛を有し、通性嫌気性、またＯＦテストで発酵性であり、好塩性で発光性をもたず、オキシダーゼ陽性、ＧＣ含量が４５．８％である等の理由から本菌株は、ビブリオ属に所属すると考えられた。そして、本菌株は、ビブリオｓｐ．Ｈ−８株として工業技術院生命工学技術研究所に寄託番号FERM P-14737として寄託されている（寄託日：平成７年１月19日）。
【００１３】
デアセチラーゼＤＡ１及びデアセチラーゼＤＡ２は、ビブリオ属に属するデアセチラーゼ生産菌を培地で培養し、培養物から得ることができる。デアセチラーゼ生産菌としては、ビブリオ属に属し、デアセチラーゼＤＡ１及びデアセチラーゼＤＡ２を産生するものであれば、いずれの菌株でも用いることができる。また、これら微生物の人工的変異方法、例えば紫外線照射、Ｘ線照射、変異誘起剤処理など、あるいは自然発生による変異株、また遺伝子操作、細胞融合による変異株でもデアセチラーゼＤＡ１またはデアセチラーゼＤＡ２を生産するものであればいずれも本発明に用いることができる。デアセチラーゼ生産能を有する菌株を選定するには、菌株の培養物を取り出し、そのデアセチラーゼ活性を測定することにより行うことができる。デアセチラーゼ生産株のうち、代表的な株としては、ビブリオｓｐ．Ｈ−８株（FERM P-14737) を挙げることができる。
【００１４】
本発明の微生物の培養には微生物の培養に一般的に用いられる培養方法を用いることができる。培地としては資化可能な炭素源、窒素源、無機物および必要な生育、生産促進物質を程よく含有する培地であれば合成培地、天然培地いずれでも使用可能である。本酵素は、誘導酵素のため炭素源として、キチン又はキチンオリゴ糖を用いることが必須である。また、そのほかに炭素源としてグルコース、澱粉、デキストリン、麦芽糖、マンノース、フラクトース、シュークロース、アラビノース、マンニトール、糖蜜などを単独または組み合わせて用いられる。更に、菌の資化能によっては炭化水素、アルコール類、有機酸なども用いられる。窒素源としては塩化アンモニウム、硝酸アンモニウム、硝酸ナトリウム、尿素、ペプトン、肉エキス、酵母エキス、乾燥酵母、コーン・スチーブ・リカー、大豆粉、カザミノ酸などが単独または組み合わせて用いられる。そのほか、食塩、塩化カリウム、硫酸マグネシウム、炭酸カルシウム、リン酸二水素カリウム、リン酸水素二カリウム、硫酸第一鉄、塩化カルシウム、硫酸マンガン、硫酸亜鉛、硫酸銅などの無機塩類や海水を必要に応じて加える。更に使用菌の生育やデアセチラーゼの生産を促進する微量成分を適当に添加することができる。
【００１５】
培養法としては、一般の培養方法が用いられるが、液体培養法、とくに深部攪拌培養法がもっとも適している。培養温度は16〜60℃、特に30〜42℃が適当であり、培養中の培地のpHはアンモニア水や炭酸アンモニウム溶液、塩酸溶液などを添加して、６〜11、特に７〜９に維持することが望ましい。液体培養で通常６〜96時間培養をおこなうと、目的物質のデアセチラーゼＤＡ１及びデアセチラーゼＤＡ２が菌体外に生成される。培養物中の生成量が最大に達したときに培養を停止する。
【００１６】
培養物からデアセチラーゼＤＡ１及びデアセチラーゼＤＡ２の分離、精製は、酵素をその培養物から単離精製するために常用される方法に従っておこなわれる。例えば培養物を濾過により培養濾液と菌体に分け、培養ろ液を硫酸アンモニウム等を用い、塩析によって沈澱物を得る。この沈澱物を溶解、透析、ゲルろ過クロマトグラフィー、イオン交換クロマトグラフィー等の手順で精製することができる。
【００１７】
以上の手順で生成されたデアセチラーゼＤＡ１は、以下の酵素化学的性質を有している。
（１）Ｎ−アセチルキトビオースあるいはＮ−アセチルキトトリオースに作用して、脱アセチル化する。
（２）至適 pH ：37℃で8.５−9.０
（３）至適温度：pH8.５で45℃
（４）pH安定性：37℃で6.０−11.0で安定
（５）熱安定性：pH8.５で50℃で１時間の処理に安定
（６）分子量：20,000−60,000（ＳＤＳ−ＰＡＧＥ法）
（７）等電点：3.３
デアセチラーゼＤＡ２は、以下の理化学的性質を有している。
（１）Ｎ−アセチルキトビオースあるいはＮ−アセチルキトトリオースに作用して、脱アセチル化する。
（２）至適 pH ：37℃で8.０−8.５
（３）至適温度：pH8.５で40℃
（４）pH安定性：37℃で7.０−11.0で安定
（５）熱安定性：pH8.５で40−55℃℃で１時間の処理に安定
（６）分子量：20,000−60,000（ＳＤＳ−ＰＡＧＥ法）
（７）等電点：3.５
以上の酵素化学的性質及び後述の酵素活性について、本発明のデアセチラーゼと公知のデアセチラーゼとを比較したところ、Ｎ−アセチルグルコサミンのオリゴマーであるＮ−アセチルキトビオース及びＮ−アセチルキトトリオースに選択的に作用する点、及び、酵素活性がタンパク質１mg当たり、10ユニット以上である点で、公知のデアセチラーゼと明らかに相違する。そこで、デアセチラーゼＤＡ１及びデアセチラーゼＤＡ２を新規酵素と認定した。
【００１８】
本発明の酵素は、医薬品等の合成中間体として有用なＮ−アセチルキトビオース及びＮ−アセチルキトトリオースの脱アセチル化物の製造に用いることができる。
【００１９】
【実施例】
以下、実施例により本発明を更に詳細に説明するが、本発明はこれに限定されるものではない。
［実施例１］
キチン粉末５g/L 、ペプトン５g/L 、酵母エキス１g/L 、リン酸第二鉄0.01g/L 、天然海水1000mlの組成を有する前培養培地（殺菌前 pH 7.５) 30mlを300ml フラスコに加え、ビブリオｓｐ．Ｈ−８株を種菌として植菌し、37℃、24時間培養した。このようにして得られた前培養液を 100Ｌ容量の発酵槽中の上記組成と同一組成の培地30Ｌに５％v/v の割合で植菌し、30℃で通気攪拌方式（回転数300rpm, 通気量0.7vvm) により18時間培養を行った。この際、pHは7.８になるように調整しながら培養した。
［実施例２］
実施例１で得られた培養液30Ｌを1000rpm で遠心分離し、培養上清を得た。上清を硫酸アンモニウムで塩析し（80％飽和）、沈澱物をろ過後、50mM リン酸ナトリウム緩衝液（pH7.０）２Ｌに懸濁し、そのろ液を粗酵素液とした。次に、50ＦＴ-Ｃ-110透析チューブ（三光純薬製) に上記粗酵素を入れ、50mM リン酸ナトリウム緩衝液40Ｌを透析液として透析を行った。透析した酵素懸濁液をＤＥＡＥセファロースＦＦ（ファルマシアバイオテク社製）カラムを用いて、NaClのグラジェントによりデアセチラーゼ活性画分を溶出した。溶出画分はさらにブチルセファロース４ＦＦ（ファルマシアバイオテク社製) に吸着させ、硫酸アンモニウムのグラジェントにより溶出した。デアセチラーゼ活性画分は２つに分かれた。２つの画分をそれぞれセファクリルＳ-200ＨＲ（ファルマシアバイオテク社製）を用いてゲルろ過クロマトグラフィーを行った。その結果、精製デアセチラーデＤＡ１を25mg及び精製デアセチラーゼＤＡ２を７mg得ることができた。
［実施例３］
デアセチラーゼＤＡ１及びデアセチラーゼＤＡ２の酵素活性の測定には、亜硝酸−インドール反応（Z. Dische, E. Borenferund, J. Biol. Chem., 184, 517 (1950)) を用いた。なお、デアセチラーゼ１ユニットは１分間に１μmol のグルコサミンを遊離する活性を有する量とする。上記の測定法に従ってデアセチラーデＤＡ１及びデアセチラーゼＤＡ２の酵素活性を測定した。その結果、タンパク質１mgあたり、デアセチラーゼＤＡ１の活性は、32.5ユニットまた、デアセチラーゼＤＡ２の活性は、35ユニットであった。
【００２０】
【発明の効果】
本発明は、高活性で、かつ、Ｎ−アセチルグルコサミンのオリゴマーに選択的に作用する新規なデアセチラーゼを提供する。[0001]
[Industrial application fields]
The present invention relates to novel deacetylase DA1 and deacetylase DA2 produced by microorganisms belonging to the genus Vibrio. The enzyme of the present invention is important for producing deacetylated products of N-acetylchitobiose and N-acetylchitotriose which are useful as synthetic intermediates for pharmaceuticals and the like.
[0002]
[Prior art]
The enzyme that deacetylates N-acetylglucosamine or its oligomer has not been studied so far. Recently, Fujishima et al. (Biosci. Biotech. Biochem., Vol 58 193-195 1994) (Japanese Patent Laid-Open No. 4-349884) It has been reported.
[0003]
However, the above enzyme is an intracellular enzyme, and its activity per mg of purified enzyme protein is very weak at 0.11 unit, and it selectively acts only on N-acetylglucosamine in terms of substrate specificity. That is, an enzyme that is produced outside the cell and has a high activity of 10 units or more per 1 mg of purified enzyme protein and that selectively acts on N-acetylchitobiose or N-acetylchitotriose is known. It is not done.
[0004]
[Problems to be solved by the invention]
An object of the present invention is to provide a novel deacetylase that selectively acts on N-acetylchitobiose or N-acetylchitotriose, which is an oligomer of N-acetylglucosamine, and has higher activity than conventional deacetylases. It is in.
[0005]
[Means for Solving the Problems]
As a result of searching for a marine bacterium-derived deacetylase to solve the above problems, the present inventors have found that the H-8 strain belonging to the genus Vibrio has a high deacetylase activity, and N-acetylchitobiose. Alternatively, the inventors have found that a deacetylase that selectively acts on acetylchitotriose is produced, and the present invention has been completed.
[0006]
That is, the present invention is deacetylase DA1 or deacetylase DA2 having the following enzymatic chemistry.
A. Deacetylase DA1
(1) Deacetylation by acting on N-acetylchitobiose or N-acetylchitotriose.
(2) Optimum pH: 8.5-9.0 at 37 ° C
(3) Optimal temperature: 45 ° C at pH 8.5
(4) pH stability: stable at 6.0-11.0 at 37 ° C. (5) thermal stability: stable at pH 8.5 for 1 hour treatment at 50 ° C. (6) molecular weight: 20,000-60,000 (SDS-PAGE method) )
(7) Isoelectric point: 3.3
B. Deacetylase DA2
(1) Deacetylation by acting on N-acetylchitobiose or N-acetylchitotriose.
(2) Optimum pH: 8.0-8.5 at 37 ° C
(3) Optimal temperature: 40 ° C at pH 8.5
(4) pH stability: stable at 7.0-11.0 at 37 ° C. (5) thermal stability: stable at 1 hour treatment at pH 8.5 at 50 ° C. (6) molecular weight: 20,000-60,000 (SDS-PAGE method) )
(7) Isoelectric point: 3.5
The present invention also relates to a method for producing deacetylase DA1 or deacetylase DA2, which comprises culturing a microorganism having the ability to produce deacetylase DA1 or deacetylase DA2 belonging to the genus Vibrio, and collecting deacetylase deDA1 or deacetylase DA2 from the culture. It is.
[0007]
Hereinafter, the present invention will be described in detail.
The H-8 strain was isolated from the bottom mud collected at Lake Hamana, Shizuoka Prefecture.
The bacteriological properties of the H-8 strain are shown below. As the medium, marine agar (Bacto Marine Agar 2216) (Difco) or marine broth (Bifo Marine Broth 2216) (Difco) was used. These strains were inoculated into these media, cultured at 30 ° C. for 24-48 hours, and observed for morphology using an optical microscope and a transmission electron microscope. Moreover, since this strain requires sodium chloride, the biochemical property test was performed by preparing a medium using 75% artificial seawater or adding 3% sodium chloride to the medium.
(1) Form (under aerobic conditions)
Cell shape and size: Aspergillus without bending, 0.7-1.1 × 1.3-1.8 μm
Presence / absence of cell pluripotency: No motility: Yes Flagellar state: Polar hair, when cultured in a solid medium, peripheral flagella are observed around the cells.
[0008]
Swarming: Presence of spores: None Gram stainability: Negative (2) Growth state in each medium Marine agar plate culture: Grows well, colonies grow in a convex circle, all-edge wet light, opaque milky white The surface is smooth.
[0009]
Marine agar slope culture: good growth, filamentous, milky white marine broth culture: good growth, the culture solution becomes cloudy uniformly.
Marine broth gelatin puncture culture: liquefy (after 1 week).
Litmus milk: digested (3) Physiological properties Reduction of nitrate: Formation of reducing indole: Produced.
[0010]
Production of hydrogen sulfide: Not produced in TSI medium.
Starch decomposition: Hydrolyzes.
Use of citric acid: Use Simons medium.
: Use Cristensen medium.
Use of inorganic nitrogen source: Use.
[0011]

[0012]

Based on the above bacteriological properties, as a result of comparison with known strains according to the classification criteria of the Virgin's Manual of Determinative Bacteriology 8th edition, it is a Gram-negative bacilli and has polar flagellum, facultative This strain is considered to belong to the genus Vibrio because it is anaerobic, fermentable by OF test, halophilic, non-luminescent, oxidase positive, GC content is 45.8%, etc. It was. And this strain is Vibrio sp. It has been deposited as the H-8 stock at the National Institute of Biotechnology, Depository No. FERM P-14737 (Deposit date: January 19, 1995).
[0013]
Deacetylase DA1 and deacetylase DA2 can be obtained from a culture obtained by culturing a deacetylase-producing bacterium belonging to the genus Vibrio in a medium. Any strain can be used as long as it belongs to the genus Vibrio and produces deacetylase DA1 and deacetylase DA2. In addition, artificial mutation methods for these microorganisms, such as ultraviolet irradiation, X-ray irradiation, treatment with mutagenic agents, naturally occurring mutant strains, and genetically engineered and cell fusion mutant strains that produce deacetylase DA1 or deacetylase DA2. Any can be used in the present invention. Selection of a strain having the ability to produce deacetylase can be performed by taking out a culture of the strain and measuring its deacetylase activity. Among the deacetylase-producing strains, representative strains include Vibrio sp. And H-8 strain (FERM P-14737).
[0014]
For culturing the microorganism of the present invention, a culture method generally used for culturing microorganisms can be used. As a medium, any medium can be used as long as it contains moderately assimilable carbon sources, nitrogen sources, inorganic substances, and necessary growth and production promoting substances. Since this enzyme is an inducing enzyme, it is essential to use chitin or chitin oligosaccharide as a carbon source. In addition, glucose, starch, dextrin, maltose, mannose, fructose, sucrose, arabinose, mannitol, molasses, etc. may be used alone or in combination as a carbon source. Furthermore, hydrocarbons, alcohols, organic acids, and the like are also used depending on the ability of assimilation of bacteria. As the nitrogen source, ammonium chloride, ammonium nitrate, sodium nitrate, urea, peptone, meat extract, yeast extract, dry yeast, corn stew liquor, soy flour, casamino acid and the like are used alone or in combination. In addition, salt, potassium chloride, magnesium sulfate, calcium carbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ferrous sulfate, calcium chloride, manganese sulfate, zinc sulfate, copper sulfate and other inorganic salts and seawater are required. Add as appropriate. Furthermore, trace components that promote the growth of the bacteria used and the production of deacetylase can be appropriately added.
[0015]
As a culture method, a general culture method is used, but a liquid culture method, particularly a deep stirring culture method is most suitable. The culture temperature is suitably 16 to 60 ° C., particularly 30 to 42 ° C. The pH of the medium during the culture is maintained at 6 to 11, particularly 7 to 9 by adding aqueous ammonia, ammonium carbonate solution, hydrochloric acid solution or the like. It is desirable to do. When culturing is usually carried out for 6 to 96 hours in liquid culture, the target substances deacetylase DA1 and deacetylase DA2 are produced outside the cells. The culture is stopped when the production amount in the culture reaches the maximum.
[0016]
Separation and purification of deacetylase DA1 and deacetylase DA2 from the culture is performed according to a method commonly used for isolating and purifying the enzyme from the culture. For example, the culture is separated into a culture filtrate and cells by filtration, and the culture filtrate is ammonium sulfate or the like, and a precipitate is obtained by salting out. This precipitate can be purified by procedures such as dissolution, dialysis, gel filtration chromatography, ion exchange chromatography and the like.
[0017]
The deacetylase DA1 produced by the above procedure has the following enzyme chemical properties.
(1) Deacetylation by acting on N-acetylchitobiose or N-acetylchitotriose.
(2) Optimum pH: 8.5-9.0 at 37 ° C
(3) Optimal temperature: 45 ° C at pH 8.5
(4) pH stability: stable at 6.0-11.0 at 37 ° C. (5) thermal stability: stable at pH 8.5 for 1 hour treatment at 50 ° C. (6) molecular weight: 20,000-60,000 (SDS-PAGE method) )
(7) Isoelectric point: 3.3
Deacetylase DA2 has the following physicochemical properties.
(1) Deacetylation by acting on N-acetylchitobiose or N-acetylchitotriose.
(2) Optimum pH: 8.0-8.5 at 37 ° C
(3) Optimal temperature: 40 ° C at pH 8.5
(4) pH stability: stable at 7.0-11.0 at 37 ° C (5) thermal stability: stable at treatment at 40-55 ° C for 1 hour at pH 8.5 (6) molecular weight: 20,000-60,000 (SDS -PAGE method)
(7) Isoelectric point: 3.5
When the deacetylase of the present invention and the known deacetylase were compared with respect to the above enzyme chemical properties and the enzyme activity described below, N-acetylchitobiose and N-acetylchitotriose, which are oligomers of N-acetylglucosamine, were selected. It is clearly different from known deacetylases in that it acts on the surface and the enzyme activity is 10 units or more per mg of protein. Therefore, deacetylase DA1 and deacetylase DA2 were certified as new enzymes.
[0018]
The enzyme of the present invention can be used for the production of N-acetylchitobiose and N-acetylchitotriose deacetylated compounds useful as synthetic intermediates for pharmaceuticals and the like.
[0019]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to this.
[Example 1]
30 ml of pre-culture medium (pH 7.5 before sterilization) having the composition of 5 g / L chitin powder, 5 g / L peptone, 1 g / L yeast extract, 0.01 g / L ferric phosphate and 1000 ml natural seawater in a 300 ml flask In addition, Vibrio sp. The H-8 strain was inoculated as an inoculum and cultured at 37 ° C. for 24 hours. The preculture solution thus obtained was inoculated at a rate of 5% v / v into 30 L of a medium having the same composition as the above composition in a 100 L capacity fermenter, and aerated and stirred at 30 ° C. The culture was performed for 18 hours with an aeration rate of 0.7 vvm. At this time, the culture was performed while adjusting the pH to 7.8.
[Example 2]
30 L of the culture solution obtained in Example 1 was centrifuged at 1000 rpm to obtain a culture supernatant. The supernatant was salted out with ammonium sulfate (80% saturation), the precipitate was filtered, suspended in 2 L of 50 mM sodium phosphate buffer (pH 7.0), and the filtrate was used as a crude enzyme solution. Next, the crude enzyme was placed in a 50FT-C-110 dialysis tube (manufactured by Sanko Junyaku Co., Ltd.) and dialyzed using 40 L of 50 mM sodium phosphate buffer as a dialysis solution. The deacetylase activity fraction was eluted from the dialyzed enzyme suspension using a DEAE Sepharose FF (Pharmacia Biotech) column with a NaCl gradient. The eluted fraction was further adsorbed onto butyl sepharose 4FF (Pharmacia Biotech) and eluted with an ammonium sulfate gradient. The deacetylase activity fraction was divided into two. The two fractions were subjected to gel filtration chromatography using Sephacryl S-200HR (Pharmacia Biotech). As a result, 25 mg of purified deacetylase DA1 and 7 mg of purified deacetylase DA2 could be obtained.
[Example 3]
A nitrite-indole reaction (Z. Dische, E. Borenferund, J. Biol. Chem., 184, 517 (1950)) was used to measure the enzyme activities of deacetylase DA1 and deacetylase DA2. One unit of deacetylase is an amount having an activity of releasing 1 μmol of glucosamine per minute. The enzyme activities of deacetylase de DA1 and deacetylase DA2 were measured according to the above measurement method. As a result, the activity of deacetylase DA1 was 32.5 units and the activity of deacetylase DA2 was 35 units per 1 mg of protein.
[0020]
【The invention's effect】
The present invention provides a novel deacetylase that is highly active and selectively acts on oligomers of N-acetylglucosamine.

Claims (3)

Deacetylase DA1 having the following enzymatic chemistry .
(1) Deacetylation by acting on N-acetylchitobiose or N-acetylchitotriose.
(2) Optimum pH: 8.5-9.0 at 37 ° C
(3) Optimal temperature: 45 ° C at pH 8.5
(4) pH stability: stable at 6.0-11.0 at 37 ° C (5) thermal stability: stable at 1 hour treatment at pH 8.5 at 50 ° C (6) Molecular weight: 20,000-60,000 (SDS-PAGE method)
(7) Isoelectric point: 3.3
(8) Vibrio sp. Produced by the H-8 strain (Accession Number FERM P-14737 ).
(9) Vibrio sp. The culture supernatant of H-8 strain (Accession No. FERM P-14737 ) is salted out with ammonium sulfate, and the resulting precipitate is dissolved, dialyzed, subjected to DEAE Sepharose column chromatography and eluted with NaCl gradient to deacetylase. The active fraction is obtained and further subjected to butyl sepharose column chromatography, and one of the two deacetylase active fractions eluted with an ammonium sulfate gradient is obtained by gel filtration chromatography. Deacetylase DA2 having the following enzyme chemistry.
(1) Deacetylation by acting on N-acetylchitobiose or N-acetylchitotriose.
(2) Optimal  pH : 37 At ℃ 8.0 − 8.5
(3) Optimal temperature: pH8.5 so 40 ℃
(4) pH Stability: 37 At ℃ 7.0 − 11.0 Stable
(5) Thermal stability: pH8.5 so 50 At ℃ 1 Stable in processing time
(6) Molecular weight: 20,000 − 60,000 (SDS-PAGE method)
(7) Isoelectric point: 3.5
(8) Vibrio sp. H-8 strain (Accession number) FERM P-14737 ) Produced by.
(9) Vibrio sp. H-8 strain (Accession number) FERM P-14737 ) Salting out the culture supernatant with ammonium sulfate, dissolving the resulting precipitate, dialyzing, DEAE Sepharose column chromatography NaCl The deacetylase activity fraction is obtained by elution with a gradient of 2 and then subjected to butyl sepharose column chromatography, and one of the two deacetylase activity fractions eluted with an ammonium sulfate gradient is obtained by gel filtration chromatography. Vibrio sp. The H-8 strain (Accession No. FERM P-14737 ) is cultured, and the deacetylase DA1 according to claim 1 or the deacetylase DA2 according to claim 2 is collected from the culture. Deacetylase DA1 or deacetylase DA2 Manufacturing method.